The present invention relates to adjuvant combinations comprising two or more different adjuvants. In particular the invention relates to adjuvant compositions comprising the adjuvants in aqueous media for immunization and vaccines.
The invention also relates to vaccines and immunization combination kits comprising two or more adjuvants and an antigenic substance.
Since the English doctor Edward Jenner in 1796 discovered that the infectious agency causing cowpox in cattle was able to produce immunity against smallpox in human beings without causing serious illness many efforts have been made in order to find other vaccines which can generate immunity against more or less severe diseases in animal and human beings without provoking the unpleasant, serious or fatal symptoms and reactions usually accompanying the ordinary diseases in question.
Thus, for example, tuberculosis in man has for many years been combated by vaccination with attenuated but living strains of Mycobacterium bovis (BCG vaccine). However, the efficacy of this procedure does not allways provide satisfactory resistance to human tuberculosis in every population.
Therefore, attempts have been made to isolate and use fragments or subfragments of strains of human Mycobacterium tuberculosis instead as immunogenic agent which when injected intradermally or subcutaneously in individuals would cause satisfactory immunity against infections with naturally occurring strains of human Mycobacterium tuberculosis. Thus, non-determined substances from culture filtrates as well as a few isolated molecules such as Ag85 and ESAT-6 of Mycobacterium tuberculosis have been shown to provide some degree of tuberculosis immunity.
In the future it would be desirable to have vaccines based on well-defined substances which would always create high immunity against tuberculosis and other diseases.
Unfortunately, many highly purified substances, e.g.. purified recombinant proteins, are not very immunogenic and do not generate an effective immune response protective against the real infectious disease. This fact has been recognized since the beginning of this century and it has been tried to counteract the low immunogenicity by combining the substance in question with immunogenic response potentiating agents, so-called adjuvants. A large number of such adjuvants and kind of adjuvants have been suggested but in general without any being ideal in all respects.
The present inventors have now discovered that two particular classes of adjuvants possess the capability to elicit a strong and long persisting immune response when administered in combination with an antigenic substance, even though this substance may have only poor immunogenicity per se.
Thus, the present invention relates to an adjuvant combination comprising a first adjuvant component which is a quaternary hydrocarbon ammonium halogenide of the formula NR1R2R3R4-hal, wherein R1 and R2 independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R3 and R4 independently each is a hydrocarbon group containing from 12 to 20 carbon atoms, preferably from 14 to 18 carbon atoms and hal is a halogen atom, and a hydrophobic second adjuvant component
In the formula NR1R2R3R4-hal the R1 and R2 groups may e.g. be methyl, ethyl, propyl and isopropyl, whereas R3 and R4 may be dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl nonadecyl and eicocyl groups. However, also other C12-C20 hydrocarbon groups are possible because even though the R3 and R4 groups usually and preferably are straight chain hydrocarbon groups they may in minor degree be branched having e.g. methyl and ethyl side chains. R3 and R4 may also have a minor degree of unsaturation, e.g. containing 1-3 double bonds each, but preferably they are saturated alkyl groups. R3 and R4 are preferably saturated alkyl groups containing from 14 to 18 carbon atoms.
The halogen atom xe2x80x9chalxe2x80x9d is preferably bromine or chlorine because the other halogens, fluorine and iodine, may have undesirable biochemical, physiological and injurious effects, but for some experimental purposes, where such effects can be accepted, they may also be selected.
Preferably the hydrophobic second adjuvant component is selected from the group comprising triterpenoid saponins and derivatives thereof, lipopolysaccharides (LPS) and derivatives thereof, Staphylococcus antigen A, carbohydrate coupled phospholipids, monophosphoryl lipid A (MPL-A), mineral oil , Neem oil, taxol, the squalane and squalene series of adjuvants, block co-polymer adjuvants, pleuronic bloc polymer adjuvants, and lipoglycanes.
Examples of block co-polymer adjuvants are described by e.g. Todd C. V. et al., Systematic development of a block copolymer adjuvant for trivalent influenza virus vaccine, Dev Biol Stand 1998; 92:341-51.
Amongst the hydrophobic second adjuvant components, lipophilic adjuvants, such as monophosphoryl lipids (MPL), are prefered.
The monophosphoryl lipids (MPL) are e.g. obtainable from microbial lipopolysaccharide (LPS) and are usually prepared from bacterial polysaccharides even though other microbial sources like viruses, moulds, fungi, yeasts and algae may be the source of origin for the phosphoryl lipid of choice. Suitable bacterial polysaccharides are e.g. described in xe2x80x9cThe Theory and Practical Applications of adjuvantsxe2x80x9d1), chapter thirteen, pp. 287-313, Ed. by D. E. S. Stewart-Tull, 1995, John Wiley Sons Ltd., in xe2x80x9cMethods in Microbiologyxe2x80x9d2), Vol. 25, pp. 471-502, Ed. Stefan A E Kaufmann and Dieter Kabelitz, 1998, Academic Press, San Diego, Calif., USA and London, UK, and in xe2x80x9cVaccinexe2x80x9d3), vol. 15, No. 3, pp. 248-256, 1997, Elsevier Science Ltd., GB.
Also, the monophosphoryl lipids derivable from the microbial polysaccharides and suitable for use in the adjuvant combinations of the present invention are described in more details in the above referrences. The preferred monophosphoryl lipid is monophosphoryl lipid A (MPL-A) which is described in 1) on pp. 289-292. in 2) on pp. 483-484, and in 3) on page 252, column 2. The most preferred MPL-A is designated 3-O-deacylated monophosphoryl lipid A. However, also other derivatives of the MPL-A""s may be applicable.
The adjuvant combination of the present invention may preferably be in the form of:
a) an aqueous composition comprising the quaternary hydrocarbon ammonium halogenide of the formula NR1R2R3R4-hal, wherein R1 and R2 independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R3 and R4 independently each is a medium chain length hydrocarbon group containing 12 to 20 carbon atoms and hal is a halogen atom, and
b) an aqueous composition comprising the hydrophobic second adjuvant component.
The aqueous media in these aqueous compositions may be any suitable aqueous solvent. However, formation of useful possible micelle structures appears to be sensitive to anions, like phosphate and sulphate ions. Thus, it is preferred that the adjuvant compositions of the Inventions are formed in the absence or low levels of such ions.
The aqueous adjuvant compositions may be prepared by any suitable process or procedure, e.g. as described further on in the detailed part of this specification.
If expedient, the different adjuvant compositions may be combined into one single composition either as a stock composition or immediately before use.
The invention concerns also a kit for immunization, said kit comprising a first adjuvant component which is a quaternary hydrocarbon ammonium halogenide of the formula NR1R2R3R4-hal, wherein R1 and R2 independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R3 and R4 independently each is a hydrocarbon group containing from 12 to 20 carbon atoms, preferably from 14 to 18 carbon atoms, and hal is a halogen atom, and a hydrophobic second adjuvant component and an antigenic substance.
Such kit may be presented in the form of individual containers or compartments containing the different adjuvants and the antigenic substance and any solvent necessary for effecting the immunization procedure as well as any necessary device for the performance thereof. If appropriate the adjuvants and the antigenic substance may also be combined and stocked in one single container. If the adjuvants and the antigenic substance each is contained in a separate container they may be mixed in any order before use. For some applications it may be advantageous, however, to mix the adjuvants and the antigenic substances in a particular order for obtaining optimum results.
In principle the antigenic substance may be any pure chemical species such as a protein or a fragment thereof or artificial mixtures prepared of such species. But it can also be any naturally occuring mixture of chemical species such as e.g. a cell homogenate or fractions thereof, a culture filtrate from microorganisms or cell tissues from multicellular organisms, e.g. higher animals.
Specifically the antigenic substance may be derived from a culture of metabolizing Mycobacterium tuberculosis, Mycobacterium bovis and other environmental Mycobacteria such as e.g. Mycobacterium avium. A particular interesting substance from the filtrate of such Mycobacteria is the ESAT-6 protein (Early Secretory Antigenic Target) which is a dominant target for cell mediated immunity in the early phase of tuberculosis in TB patients and in different animal models. It contains 95 amino acid residues and has a deduced molecular mass of approximately 10 kDa. Its immunogenicity per se is low, but in combination with the adjuvant combinations of the present invention it has turned out to be a potent candidate for provoking high and persisting immunity against tuberculosis as is demonstrated in the following detailed part of this specification.
ESAT-6 as well as many other antigens applicable in combination with the adjuvant combinations of the present invention, today can be produced artificially, e.g. synthetically or by genetic recombinant techniques.
In addition to provide immunity to diseases the adjuvant combinations of the present invention can also be used for producing antibodies against compounds which are poor immunogenic substances per se and such antibodies can be used for the detection and quantification of the compounds in question, e.g. in medicine and analytical chemistry.
Without being bound by theory it is believed that an adjuvant as DDA, which induces strong CMI (cell mediated immune) reponses, has the ability to form micelles in aqueous solutions. The lipid portion of this structure provides a matrix for the inclusion of other lipophilic compounds and the formation of composite micelles with increased adjuvant activity.
Preferred embodiments of the adjuvant combination and the immunization combination kit of the present invention are set forth in the dependent claims in the accompanying set of claims attached.
The invention will now be further described and illustrated by reference to embodiment examples of the invention and experimental examples forming the basis for the discovery of the present invention.
Animals
The studies were performed with 8 to 12 weeks old C57BL/6 (C57BL/6J, H-2b) female mice, purchased from Bomholtegaard, Ry, Denmark.
Infected animals were housed in cages contained within a laminar flow safety enclosure.
Bacteria
M. tuberculosis H37Rv was grown at 37xc2x0 C. on Lxc3x6wenstein Jensen medium or in suspension in modified Sauton medium enriched with 0.5% sodium pyruvate and 0.5% glucose.
Adjuvants
DDA-Br (Eastman Kodak, Inc., Rochester, N.Y.) is mixed into sterile water to a concentration of 2.5 mg/ml and heated to 80xc2x0 C. while stirring continuously on a magnetic stirring hotplate for 10 min and then cooled to room temperature before use.
MPL-A (MPL, Ribi Immunochem, Hamilton, Mont., USA) is mixed into sterile water to a concentration of 1 mg/ml containing 2 xcexcl triethylamine. The mixture is heated in a 65-70xc2x0 C. water bath for 30 seconds and then sonicated for 30 seconds. The heating and sonicating procedure is repeated twice. The solution is stored at 4xc2x0 C. until use.
On the morning of immunization the antigen is mixed with saline and MPL-A (in the following abbreviated to MPL) is added. Then DDA-Br (in the following abbreviated to DDA) is added and the suspension is mixed on a vortex mixer.
Immunization
Mice were immunized three times with two weeks intervals subcutaneously (sc) in the back with the experimental vaccines which contained either 50 xcexcg ST-CF/dose or 10-50 xcexcg ESAT-6/dose emulsified in DDA (250 xcexcg/dose, Eastman Kodak, Inc., Rochester, N.Y.) with or without 25 xcexcg monophosphoryl lipid A (MPL, Ribi Immunochem, Hamilton, Mont., USA) in a volume of 0.2 ml.
A single dose of BCG Copenhagen 1331 (5xc3x97104 cfu (colony forming units)) was injected subcutaneously at the base of the tail.
Experimental Infections
Intravenous (iv) infections were administered via the lateral tail vein with an inoculum of 5xc3x97104 M. tuberculosis (H37Rv) suspended in phosphate buffered saline (PBS) in a volume of 0.1 ml. The mice were sacrificed two weeks later.
Respiratory infections (ri) of the animals with M.tuberculosis (Erdman) were administered by the aerosol route with an inoculum of 5xc3x97106/ml. Six weeks later the mice were sacrificed.
Bacterial numbers in the liver, spleen or lung were determined by double serial 3 fold dilutions of individual whole organ homogenates on Middlebrook 7H11 medium. Organs from the BCG vaccinated animals were grown on medium supplemented with 2 xcexcg 2-thiophene-carboxylic acid hydrazide (TCH). Colonies were counted after 3 weeks of incubation at 37xc2x0 C. The protective efficacies are expressed as means of the bacterial counts in immunized mice after subtraction of the adjuvant control obtained from 5 animals/group.
Mycobacterial Antigens
Short-term culture filtrate (ST-CF) was produced as described previously {Andersen P., Askgaard D., Ljungqvist L., Bennedsen J., and Heron I., Proteins released from Mycobacterium tuberculosis during growth. Infect. Immun. 59: 1905-1910, 1991}. Briefly, M tuberculosis (8xc3x97106 CFU/ml) were grown in modified Sauton medium without Tween 80 on an orbital shaker for 7 days. The culture supernatants were sterile filtered and concentrated on an Amicon YM3 membrane (Amicon, Danvers, Mass.)
Recombinant ESAT6 was prepared by Brandt et al. {Brandt L., Oettinger T., and Andersen P., Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacterium tuberculosis. J. Immunol. 157:3527-3533, 1996} The LPS content in the preparations was measured by the LAL test to be below 0.3 ng/xcexcg protein and this concentration had no influence on cellular activity. The protein was kept at xe2x88x9280xc2x0 C. until use.
Lymphocyte Cultures
Lymphocytes from spleens were obtained as described previously {Andersen P., Askgaard D., Ljungqvist L., Bentzon M. W., and Heron I., T-cell proliferative response to antigens secreted by Mycobacterium tuberculosis. Infect. Immun. 59: 1558-1563, 1991}. Blood lymphocytes were purified on density medium. Cells pooled from 3-5 mice in each experiment were cultured in microtiter wells (96 well, Nunc, Roskilde, Denmark) containing 2xc3x97105 cells in a volume of 200 xcexcl RPMI 1640 supplemented with 2-mercaptoethanol, Penicillin-Streptomycin, glutamine and 5% (vol/vol) FCS (foetal calf serum). ST-CF and the preparations of ESAT-6 were used in various concentrations (2-20 mg/ml) in the cultures. Culture filtrate fractions were used at 5 mg/ml. Based on previous dose-response investigations, purified mycobacterial antigens and the peptides were all used at 5-10 mg/ml. Con A at a concentration of 1 mg/ml was used in all experiments as a positive control for cell viability. All preparations were tested in cell cultures and found to be non-toxic at the concentrations used in the present study. Supernatants were harvested from parallel cultures for the investigation of cytokines after 72 h of incubation.
IFN-xcex3 ELISA
Microtiter plates (96 well, maxisorb, Nunc) were coated with monoclonal Hamster anti-murine IFN-xcex3 (Genzyme, Cambridge, Mass.) in PBS at 4xc2x0 C. Free bindings site were blocked with 1% (wt/vol) BSA/0.05% Tween 20. Culture supernatants were tested in triplicates and IFN-xcex3 was detected by biotin-labeled rat anti-murine monoclonal antibody (clone XMG1.2, Pharmingen, San Diego, Calif.). Recombinant IFN-xcex3 (Pharmingen) was used as a standard.
ELISPOT Technique
In this assay microtiter plates (96 well, maxisorb) were coated with 2.5 mg/ml of monoclonal hamster anti-murine IFN-xcex3 (Genzyme). Free binding sites were blocked with bovine serum albumin followed by washing with PBS/0.05% Tween 20. Analyses were always conducted on cells pooled from three mice. Cells were stimulated with optimal concentrations of antigen in modified RPMI 1640 for 18-22 h and subsequently cultured without antigen for 7 h directly in the ELISPOT plates. The cells were removed by washing and the site of cytokine secretion detected by biotin-labeled rat anti-murine IFN-xcex3 monoclonal antibody (clone XMG1.2, Pharmingen) and phosphatase-conjugated streptavidin (Jackson ImmunoResearch Lab., Inc., PA.). The enzyme reaction was developed with BCIP (Sigma). Blue spots were counted microscopically. The relationship between the number of cells per well and the number of spot was linear in concentrations 2xc3x97105xe2x88x926.2xc3x97103 cells/well. Wells with less than 10 spots were not used for calculations.
ESAT-6 Specific IgG ELISA
ELISA plates (NUNC Maxisorp, type 96F) were coated with ESATE-6 (0.1 xcexcg/well) overnight at 4xc2x0 C. Free binding sites were blocked by 1% bovine serum albumin-PBS. Individual mice sera from 5 mice/group were analyzed in three folds dilutions. IgG (P260, diluted 1/1000, DAKO) antibody was detected by peroxidase-conjugated rabbit anti-mouse reagent. Antibody titers are expressed as reciprocal end point titer.
Statistical Methods
Mean response to individual antigens were compared by the paired Student""s t-test. The efficacies of different vaccination protocols have been compared by one-way analysis of variance of log 10 cfu (colony forming units).